Defining the role of FUS phosphorylation in neurodegeneration

 描述(申请人提供)：融合肉瘤(FUS)是一种位于细胞核中的普遍存在的多功能RNA结合蛋白(RBP)。肌萎缩侧索硬化症(ALS)和额颞叶变性(FTLD)的亚型称为FTLD-FUS或ALS-FUS。ALS-FUS病例是由FUS基因突变引起的。在这些情况下，FUS的积累被认为是由细胞质FU的长期增加推动的，这些突变通过破坏保守的核定位信号(NLS)而减少了核进口。然而，FUS在FTLD-FUS病例中积累的原因尚不清楚。此外，目前还不清楚是什么原因导致肌萎缩侧索硬化症中的细胞质FU聚集并变得不能溶解。我们发现了一种新的机制，可以解释这两种现象。我们发现FUS可以被磷酸化，这一事件导致FUS在包括人脑星形胶质细胞和神经元在内的多个细胞中的胞浆重新分布。特别是，我们发现化学毒素引起的DNA损伤是FUS磷酸化的有效诱导因素。此外，DNA损伤还导致EWS、TAF15和TRN的细胞质积累，这模拟了FTLD-FUS病理的一个独特方面。初步证据表明，FUS的磷酸化通过破坏N-末端的一个新的核定位信号而导致细胞质中FUS数量的增加。与这一机制一致，我们发现FUS模拟磷酸在细胞质中积累并形成聚集体。这些聚集体与应激颗粒(SGS)、RNA/蛋白质颗粒的标记物共同标记，这些颗粒与ALS-FUS和其他形式的神经退行性变中包涵体的形成有关。我们的理论是，细胞质磷酸化的FU可以通过诱导颗粒隔离RNA和RNA结合蛋白，从而阻碍正常功能，通过有毒的功能获得而导致疾病。重要的是，我们发现磷酸化的FUS存在于含有FUS包裹体的人和小鼠的大脑中，这是生物化学上不能溶解的部分。最后，我们在FTLD-FUS脑中发现了-H_2AX的大量增加，这支持了DNA损伤和FUS的磷酸化是疾病发病机制的关键组成部分的观点。在这个建议中，我们重点讨论了这样的假设，即双链DNA损伤通过DNA依赖的蛋白激酶(DNA-PK)诱导FUS的磷酸化，通过损害核输入而导致FUS在细胞质中聚集。我们将通过1)定义导致FUS磷酸化的激酶和DNA损伤类型，2)确定FUS的磷酸化如何导致细胞质易位并影响功能，以及3)确定FUS磷酸化在神经退行性变中的作用来检验这一假说。这项研究将为FUS积累如何导致神经退化提供洞察，并为ALS和FTLD的药物开发策略提供信息。我们的数据表明，防止FUS形成致病RNA/应激颗粒的方法，可能通过调节DNA修复途径或DNA-PK，可能产生治疗这些毁灭性的神经退行性疾病的方法。

 DESCRIPTION (provided by applicant): Fused in Sarcoma (FUS) is a ubiquitous multifunctional RNA-binding protein (RBP) located in the nucleus. The abnormal and pathogenic aggregation of FUS in the cytoplasm of neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), termed FTLD-FUS or ALS-FUS. ALS- FUS cases are caused by mutations in the FUS gene. In these cases, the accumulation of FUS is thought to be driven by long-term increases in cytoplasmic FUS caused by mutations that decrease nuclear import through disruption of a conserved nuclear localization signal (NLS). However, it is unknown why FUS accumulates in FTLD-FUS cases. Moreover, it is unclear what causes cytoplasmic FUS in ALS to aggregate and become insoluble. We have discovered a novel mechanism that may explain both phenomena. We find that FUS can be phosphorylated and this event causes the cytoplasmic redistribution of FUS in multiple cells including human astrocytes and neurons. In particular, we find the DNA-damage, caused by chemical toxins, is a potent inducer of FUS phosphorylation. Furthermore, DNA-damage also causes cytoplasmic accumulation of EWS, TAF15, and TRN, which mimics a unique aspect of FTLD-FUS pathology. Preliminary evidence suggests that phosphorylation of FUS leads to increased amounts of FUS in the cytoplasm by disrupting a novel nuclear localization signal in the N-terminus. Consistent with this mechanism, we find that a FUS phospho-mimetic accumulates in the cytoplasm and forms aggregates. These aggregates co-label with markers of stress granules (SGs), RNA/protein granules that have been linked to the formation of inclusions in ALS-FUS and other forms of neurodegeneration. We theorize that cytoplasmic phosphorylated FUS can cause disease through a toxic gain of function by inducing granules that sequester RNA and RNA-binding proteins, impeding normal function. Importantly, we find that phosphorylated FUS occurs in the biochemically insoluble fraction of brains of human and mice with FUS inclusions. Finally, we find a large increase in -H2AX, a marker of DNA damage, in FTLD-FUS brains, supporting the idea that DNA damage and phosphorylation of FUS is a key component of disease pathogenesis. In this proposal we focus on the hypothesis that double-strand DNA damage induces phosphorylation of FUS by the DNA-dependent protein kinase (DNA-PK) causing FUS accumulation in the cytoplasm by impairing nuclear import. We will test this hypothesis by 1) Defining the kinase and types of DNA damage responsible for FUS phosphorylation, 2) Determining how phosphorylation of FUS causes cytoplasmic translocation and affects function, and 3) Determining the role of FUS phosphorylation in neurodegeneration. This research will provide insight into how FUS accumulation causes neurodegeneration and inform drug development strategies for ALS and FTLD. Our data suggest that methods to prevent FUS from forming pathogenic RNA/stress granules, potentially through modulation of the DNA- repair pathway or DNA-PK, may yield treatments for these devastating neurodegenerative diseases.